Chromatin serves an essential cellular function. Defects in chromatin and factors that modulate its structure are associated with a number of human diseases including cancer. Studying chromatin assembly on nascent DNA produced during DNA replication offers the opportunity to investigate how chromatin structure is established and maintained. Nevertheless, the analysis of chromatin assembly at the replication fork is confounded by the highly dynamic nature of the process. We have found that lagging strand synthesis and nucleosomes impact each other. In this project, we use Okazaki fragments as a tool to interrogate chromatin assembly pathways and gain a detailed picture of DNA replication. This project has three aims: Aim 1. We will analyze purified Okazaki fragments from budding yeast to test which histone chaperones are load nucleosomes on the lagging strand. We will explore the role of ATP dependent chromatin remodeling enzymes and test if nucleosome repositioning occurs as histones are being deposited. In addition, we will investigate how the predominant lagging strand polymerase, polymerase , interacts with nucleosomes to establish the ends of Okazaki fragments. These will be the first experiments ever to systematically analyze eukaryotic Okazaki fragments. Aim 2. We will map Okazaki fragments by deep sequencing to interrogate DNA replication patterns across the genome. We will map sites of initiation at replication origins as well as sites of termination genome wide. In addition, we will determine the firing characteristics of each replication origin allowing us to tell when, how often, and for how long a particular origin is active. Compiling our data, we will produce the most detailed map of DNA replication for any eukaryote, allowing fundamental aspects of genomic replication to be understood. Aim 3. We will test if the properties of Okazaki processing and replication dynamics observed in budding yeast are conserved in other eukaryotes. Using our experience with budding yeast, we will develop methods to precisely characterize Okazaki fragments in S. pombe. We will define replication origins and the dynamics of DNA replication across the genome. In addition, we will test if Okazaki fragments from S. pombe are influenced by nucleosome positions and chromatin structure. Our interdisciplinary work will address key problems that were previously intractable. These studies will be directly relevant to may research programs and will translate into a better understanding of the biological processes fundamental to maintaining genomic integrity and chromatin states.